Vector constructs

ABSTRACT

Improved vector constructs useful in the expression of double-stranded RNA are provided. The constructs are particularly useful for expression of double-stranded RNA in vitro and in vivo.

RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. 119 of Great Britainapplication number GB 0012233.3, filed May 19, 2000.

FIELD OF THE INVENTION

The invention relates to improved vector constructs for use in theexpression of double-stranded RNA, particularly for use in theexpression of double-stranded RNA in vitro and in vivo.

BACKGROUND OF THE INVENTION

Since the advent of double-stranded RNA inhibition (RNAi) as a tool forcontrolling gene expression, as described in WO 99/32619 and WO00/01846, there has been recognized a need for specialized vectorsdesigned for the production of double-stranded RNA (dsRNA).

Cloning vectors designed to produce high levels of dsRNA have beenpreviously described by Plaetinck et al. (WO 00/01846) and Timmons etal. Nature, 395:854 (1998). These vectors generally contain a multiplecloning site (MCS) into which target DNA fragments can be cloned flankedby two opposable transcriptional promoters. Essentially, these threecomponents (Promoter 1, MCS and Promoter 2) make up the entire system.In the appropriate expression system, the DNA cloned into the MCS may betranscribed in both directions, leading to the production of twocomplementary RNA strands.

A disadvantage of the known systems is that not only the cloned fragmentis transcribed. Read-through of the RNA polymerase will result intranscription of the entire vector, and this also in both directions. Asonly transcription of the cloned DNA fragment will result in activedsRNA for RNAi purposes, transcription of the vector part results inuseless, inefficient RNA. More specifically, 80% of these transcriptscan be considered as non-specific and thus non-effective.

The large amounts of non-specific RNA generated by the prior art plasmidand expression systems results in some undesirable side effects. First,in RNAi protocols based on introduction of dsRNA into C. elegans via afood organism such as E. coli which expresses the dsRNA (see WO00/01846), large RNA strands are considered to be toxic for the foodorganism. As a result, high amounts of RNA accumulating in E. coli causea significant part of the population to die.

Second, and probably more important, is the reduction of inhibitionpotential. The presence of large amounts of non-specific dsRNA causes acompetitive environment for the specified sequences. The potential ofthe template-specified dsRNA sequences to inhibit the targeted proteinexpression in, for instance, C. elegans cells is reduced by the presenceof these large non-specific regions. Such an inhibition by non-specificdsRNA has also been shown in Drosophila by Tushl et al., Genes &Development 13:3191-3197 (1999). Not only the potential to inhibit geneexpression is affected, but also the amount of specific dsRNA producedis limited.

Third, transcription of the vector backbone part, more particularlytranscription of the origin of replication and related structures,results in plasmid instability and plasmid reorganisation, leading toreduced production of dsRNA. This relatively low concentration ofeffective dsRNA in turn leads to inefficient RNAi.

To conclude, the previously described vectors have followingshortcomings: they are toxic to the feeding organism, a greaterproportion of the transcripts produced are non-specific, the inhibitorypotential of the dsRNA is reduced by the presence of non-specificregions, a high incidence of plasmid reorganizations and loss of plasmidfrom the feeding organism. It is therefore an object of the presentinvention to provide improved vectors for the production of dsRNA whichavoid the disadvantages of the prior art vectors.

Vectors for use in the in vitro synthesis of RNA transcripts, forexample the production of RNA probes, have been known and commonly usedin the art for some time (see for example F. M. Ausubel et al. (eds.),Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (1994);Jendrisak et al, Vectors for in vitro production of RNA copies of eitherstrand of a cloned DNA sequence, U.S. Pat. No. 4,766,072). In standardin vitro transcription protocols the problem of read-throughtranscription of vector sequences is generally avoided by linearizingthe transcription vector at restriction site positioned at the 3′ end ofthe desired transcript. However, this solution is not appropriate for invivo transcription or for the production of dsRNA where it is importantthat the template is transcribed in both directions.

SUMMARY OF THE INVENTION

The inventors now propose a novel solution to the problems encounteredwith the prior art vectors for the production of dsRNA, based on the useof transcription terminators. Generally the solution consists of the useof at least one transcription terminator operably linked to at least onepromoter, wherein the terminator stops the transcription initiated bythe promoter. Any DNA fragment inserted between the 3′ end of thepromoter and the 5′ end of the terminator will then be transcribed,without the unwanted transcription of the vector backbone.Preferentially the vector consists of two promoters and two terminators,as further described below.

Therefore, in accordance with a first aspect of the invention there isprovided a DNA construct comprising two opposable promoters flanking aninter-promoter region, the construct further comprising at least onetranscription terminator positioned transcriptionally downstream of oneof the said promoters. In particular, the invention provides for: a DNAconstruct comprising:

a) a first promoter and

b) a second promoter, in which the first and second promoter are inopposite orientation to each other and define:

c) an inter-promoter region positioned downstream of the 3′ end of thefirst promoter and downstream of the 3′ end of the second promoter; andwhich DNA construct further comprises:

d) at least one cloning site positioned in the inter-promoter region;and

e) a first transcription terminator, positioned (as seen from the 3′ endof the first promoter) downstream of the first promoter and downstreamof the at least one cloning site, wherein the first transcriptionterminator is operably linked to the first promoter.

The inter-promoter region can also further be defined as: the DNA regionbetween the 3′ end of the first promoter and the 3′ end of the secondpromoter, and which is downstream of the first promoter, and which isdownstream of the second promoter, and which preferably does notcontains the 5′ end of the first promoter and of the second promoter.The opposable first promoter and second promoter drive expressiondirectional from their 5′ ends to their 3′ ends starting transcriptiondownstream of their 3′ ends, thus providing transcription of bothstrands of any nucleotide sequence(s) present in the inter-promoterregion.

The two promoters present in the DNA construct of the invention may beidentical or they may be different and may be of essentially any type.The precise nature of the promoters used in the construct may bedependent on the nature of the expression system in which the constructis expected to function (e.g. prokaryotic vs eukaryotic host cell).Bacteriophage promoters, for example the T7, T3 and SP6 promoters, arepreferred for use in the constructs of the invention, since they provideadvantages of high level transcription which is dependent only onbinding of the appropriate RNA polymerase. Each of these promoters canindependently be chosen. The phage promoters can also function in a widevariety of host systems, i.e. both prokaryotic and eukaryotic hosts,provided that the cognate polymerase is present in the host cell.

The arrangement of two “opposable” promoters flanking an inter-promoterregion such that transcription initiation driven by one of the promotersresults in transcription of the sense strand of the inter-promoterregion and transcription initiation driven by the other promoter resultsin transcription of the antisense strand of the inter-promoter region isan arrangement well known in the art, for example, in the pGEM7 seriesof vectors from Promega Corp., Madison Wis., USA.

The DNA constructs of the invention differ from those of the prior artbecause of the presence of at least one transcription terminatorpositioned transcriptionally downstream of one of the promoters. Thetranscription terminator may be uni- or bi-directional, the choice ofuni- vs bi-directional terminators being influenced by the positioningof the terminator(s) within or outside the inter-promoter region, asexplained below. The terminator may be of prokaryotic, eukaryotic orphage origin. Bacteriophage terminators, for example T7, T3 and SP6terminators, are particularly preferred. The only requirement is thatthe terminator must be capable of causing termination of transcriptioninitiating at the promoter relative to which it is transcriptionallydownstream. In practice, these means that the promoter and terminatormust form a ‘functional combination’, i.e. the terminator must befunctional for the type of RNA polymerase initiating at the promoter. Byway of example, a eukaryotic RNA pol II promoter and a eukaryotic RNApol II terminator would generally form a functional combination. Theselection of a functional combination is particularly important wherebacteriophage promoters and terminators are to be used in the constructsof the invention, since the phage promoters and terminators are bothpolymerase-specific. To form a functional combination both the promoterand the terminator should be specific for the same polymerase, e.g. T7promoter and T7 terminator, T3 promoter and T3 terminator etc.

In one embodiment, the DNA construct of the invention may comprise asingle transcription terminator, positioned (as seen from the 3′ end ofthe first promoter) downstream of the first promoter and downstream ofthe at least one cloning site, wherein the first transcriptionterminator is operably linked to the first promoter, wherein the singletranscription terminator is positioned in the inter-promoter region.

In an alternative arrangement, the DNA construct comprises a singletranscription terminator positioned outside of the inter-promoterregion. In a still further embodiment, the DNA construct may comprisetwo transcription terminators, each one of which is positionedtranscriptionally downstream of one of the two promoters. In thisarrangement, one or both of the terminators may be positioned within theinter-promoter region. These various embodiments of the DNA constructsof the invention will be more fully described below, with reference tothe accompanying drawings. The position of a first transcriptionterminator outside the inter-promoter region may also be further definedas, i.e. such that a first transcription terminator is positioned (asseen from the 3′ end of the first promoter) downstream of the firstpromoter, downstream of the at least one cloning site, and downstream ofthe 5′ end of the second promoter.

The position of a second transcription terminator outside theinter-promoter region may also be further defined as, i.e. such that asecond transcription terminator positioned (as seen from the 3′ end ofthe second promoter) downstream of the second promoter, downstream ofthe at least one cloning site, and downstream of the 5′ end of the firstpromoter.

Moreover, when the terminator is not located in the inter-promoterregion, the distance between the 5′ end of the first promoter and 3′ endof the second terminator, or the distance between the 5′ end of thesecond promoter and the 3′ end of the first terminator is preferablysmall, i.e. such that the 3′ end of the first transcription terminatoris separated from the 5′ end of the second promoter by no more than 2000nucleotides, preferably no more than 1000 nucleotides, more preferablyno more than 500 nucleotides, even more preferably no more than 200nucleotides, especially preferably no more than 100 nucleotides, moreespecially preferable no more than 50 nucleotides, even more especiallypreferably no more than 20 nucleotides, particularly preferably no morethan 10 nucleotides, more particularly preferably no more than 6nucleotides.

Furthermore, when the second transcription terminator is located outsideof the inter-promoter region, preferably the 3′ end of the secondtranscription terminator is separated from the 5′ end of the firstpromoter by no more than 2000 nucleotides, preferably no more than 1000nucleotides, more preferably no more than 500 nucleotides, even morepreferably no more than 200 nucleotides, especially preferably no morethan 100 nucleotides, more especially preferably no more than 50nucleotides, even more especially preferably no more than 20nucleotides, particularly preferably no more than 10 nucleotides, moreparticularly preferably no more than 6 nucleotides.

As defined above the term ‘inter-promoter region’ refers to all of theDNA sequence between the two promoters. As explained above, in certainembodiments of the invention the transcription terminator(s) may besited within the inter-promoter region. The inter-promoter region may,advantageously, comprise a sequence of nucleotides forming a templatefor dsRNA production. The precise length and nature of this sequence isnot material to the invention. The invention further provides DNAconstructs in which the inter-promoter region comprises a cloning site.The function of the cloning site is to facilitate insertion of a DNAfragment forming a template for dsRNA production between the twopromoters. Thus, the invention provides a series of cloning vectorswhich are of general use in the construction of template vectors fordsRNA production. Also encompassed within the scope of the invention arevectors derived from the cloning vectors which have a DNA fragmentinserted into the cloning site.

The cloning site may further comprise one or more of the following:

-   -   at least one restriction site,(as known in the art), or one or        more further restriction sites, e.g. to provide a multiple        cloning site(as known in the art),    -   a stuffer DNA, e.g., flanked by at least two restriction site,        such as two BstXI restriction sites, or two XcmI restriction        sites,    -   attR1 and attR2 recombination sites,    -   a ccdB nucleotide sequence,    -   a ccdB nucleotide further comprising at least one unique        blunt-end restriction site, such as a SrfI restriction site,        and/or    -   a DNA fragment inserted in the at least one cloning site.

All of the DNA constructs provided by the invention may, advantageously,form part of a replicable cloning vector, such as, for example, aplasmid vector. In addition to the opposable promoters, inter-promoterregion and transcription terminator(s), the vector ‘backbone’ mayfurther contain one or more of the general features commonly found inreplicable vectors, for example an origin of replication to allowautonomous replication within a host cell and a selective marker, suchas an antibiotic resistance gene. The selective marker gene (e.g. theantibiotic resistance gene) may itself contain a promoter and atranscription terminator and it is to be understood that these arecompletely independent of the promoter and terminator elements requiredby the invention and are not to be taken into consideration indetermining whether a particular vector falls within the scope of theinvention.

DNA constructs according to the invention may be easily be constructedfrom the component sequence elements using standard recombinanttechniques well known in the art and described, for example, in F. M.Ausubel et al. (eds.), Current Protocols in Molecular Biology, JohnWiley & Sons, Inc. (1994), as will be appreciated by one skilled in theart from the following detailed description of the invention and theaccompanying Examples.

There follows a detailed description of DNA constructs according to theinvention, with reference to the following drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1( a) to 1(e) are schematic representations of several differentembodiments of the DNA construct according to the invention illustratingthe relative positioning of the promoter and transcription terminatorelements.

FIG. 2( a) is a schematic representation of a prior art vector includedfor comparison purposes.

FIGS. 2( b) to 2(f) are schematic representations of several furtherembodiments of the DNA construct according to the invention illustratingthe use of different cloning sites in the inter-promoter region.

FIG. 3 is a representation (plasmid map) of pGN1.

FIG. 4 is a representation (plasmid map) of pGN9.

FIG. 5 illustrates the nucleotide sequence of a fragment of plasmid pGN1(SEQ ID NO:1), annotated to show the positions of the opposable T7promoters.

FIG. 6 depicts the nucleotide sequence of the T7 transcriptionterminator (SEQ ID NO:2).

FIG. 7 illustrates the sequences of oligonucleotides oGN27 (SEQ IDNO:3), oGN28 (SEQ ID NO:4), oGN29 (SEQ ID NO:5) and oGN30 (SEQ ID NO:6)used to insert T7 transcription terminators into pGN1. The positions ofthe T7 pol terminator sequences and of various restriction sites aremarked.

FIG. 8 illustrates the nucleotide sequence of a fragment of plasmid pGN9(SEQ ID NO:7), annotated to show the positions of the opposable T7promoters (T7p) and the T7 transcription terminators (T7 term).

FIG. 9( a) is a representation (plasmid map) of pGN29; FIG. 9( b) is arepresentation (plasmid map) of pGN39; FIG. 9( c) is a representation(plasmid map) of the plasmid TopoRNAi.

FIG. 10 shows the complete nucleotide sequence of plasmid pGN9 (SEQ IDNO:8).

FIG. 11 shows the complete nucleotide sequence of plasmid pGN29 (SEQ IDNO:9).

FIG. 12 shows the complete nucleotide sequence of plasmid pGN39 (SEQ IDNO:10).

FIG. 13 shows the complete nucleotide sequence of plasmid TopoRNAi (SEQID NO:11).

FIG. 14 shows the complete sequence of plasmid pGN49A (SEQ ID NO:12).

FIG. 15 shows the complete sequence of plasmid pGN59A (SEQ ID NO:13).

FIG. 16 is a representation (plasmid map) of pGN49A.

FIG. 17 is a representation (plasmid map) of pGN59A.

DETAILED DESCRIPTION OF THE INVENTION

Referring to the Drawings, FIG. 1( a) schematically illustrates a firstDNA construct according to the invention which is a plasmid vectorcomprising two opposable promoters; a first promoter (a) and a secondpromoter (b) flanking an inter-promoter region (c), which inter-promoterregion is downstream of the 3′ of the first promoter, and down stream ofthe 3′ end of the second promoter. The first promoter and the secondpromoter may be identical or different. This embodiment comprises afirst transcription terminator (e) and a second transcription terminator(f), both of which are positioned within the inter-promoter region. Inthis embodiment, the first terminator and the second terminator arepreferentially uni-directional terminators.

A DNA fragment may be inserted in the at least one cloning site (d).Such DNA fragment is subject to transcription directed by the firstpromoter (a) and the second promoter (b) (i.e. transcription of bothstrands), resulting in the generation of two RNA fragments which maycombine to form double-stranded RNA of the inserted DNA fragment (bothin vitro and in vivo).

Any desired DNA sequence, such as a genomic DNA sequence, or a cDNAsequence or any other coding sequence, may be inserted in the at leastone cloning site. Without being limited to any specific explanation, itis assumed that when (a) and (e) form a functional combination, RNApolymerase which initiates transcription at (a) will transcribe theinter-promoter region including the at least cloning site and the DNAfragment inserted in the at least cloning site and will be terminatedwhen it reaches (e). Similarly, RNA polymerase which initiatestranscription at (b) will transcribe the inter-promoter region includingthe at least one cloning site and the DNA fragment inserted in the atleast one cloning site and will terminate when it reaches (f). Theterminators cause the RNA polymerase to pause, stop transcription andfall off the template. This prevents the unlimited transcription of thevector backbone, and reduces the unspecific transcription ofnon-essential DNA.

The inter-promoter region further comprises a sequence of nucleotidescorresponding to a target for double-stranded RNA inhibition. Thissequence is designated ‘TF’=for target fragment. It is this sequencewhich, when transcribed into dsRNA, will be responsible for specificdouble-stranded RNA inhibition of a target gene. The target fragment maybe formed from a fragment of genomic DNA or cDNA from the target gene.Its precise length and nucleotide sequence are not material to theinvention.

In the arrangement shown in FIG. 1( a) the two terminators arepositioned on either side of the TF within the inter-promoter region.Each of the terminators is positioned transcriptionally downstream ofone of the promoters, the fist terminator (e) is transcriptionallydownstream of first promoter (a) and the second terminator (f) istranscriptionally downstream of the second promoter (b). Assuming that(a) and (e) form a functional combination, as described above, RNApolymerase which initiates transcription at a) will transcribe theinter-promoter region up to and including TF and will be terminated whenit reaches (e). Similarly, RNA polymerase which initiates transcriptionat (b) will transcribe the inter-promoter region up to and including TFon the opposite strand and will terminate when it reaches (f). Theterminators cause the RNA polymerase to pause, stop transcription andfall off the template. This prevents the unlimited transcription of thevector backbone, and reduces the unspecific transcription ofnon-essential DNA.

The transcripts generated from this vector may, depending on the preciseplacement of the terminators in the vector, be almost completelyspecific dsRNAs corresponding to the TF region. Through the directplacement of the terminator sequences at the downstream end of the TFregion on both sides of the inserted DNA fragment, the amount ofmaterial transcribed is completely reduced to the template-specifiedsequences. Therefore, a higher amount of specific dsRNA is obtained.Furthermore the constructs are now also more stable, due to thenon-transcription of the vector backbone. The latter characteristic(stability), combined with the now relatively higher specifictranscription rate, provides a system adapted to synthesize higheramounts of specific short dsRNA strands. This proportionally higheramount of transcript, resulting in high concentrations of specificdsRNA, enhances the inhibitory effect in RNAi protocols. In RNAiprotocols based on expression of dsRNA in a food organism, toxicity forthe feeding organisms due to high RNA expression is brought to a minimallevel by use of this vector.

A specific example of a vector of the type illustrated in FIG. 1( a),considered by the inventors to be the optimal arrangement for RNAiapplications, is plasmid pGN9 described in the accompanying Examples.The transcriptional terminators used in pGN9 are T7 RNA polymerasespecific terminators, since the vector contains two opposable T7promoters. However, other systems could be used such as an expressionsystem based on the T3 or SP6 promoter, terminator and polymerase orother prokaryotic or eukaryotic promoters and terminators.

FIG. 1( b) illustrates schematically a further DNA construct accordingto the invention which is a plasmid vector comprising two opposablepromoters (a) and (b) flanking an inter-promoter region (c). This vectoralso comprises two transcription terminators (e) and (f) but in thisarrangement the two terminators are positioned outside of theinter-promoter region, in fact the terminator elements now flank the twopromoters. The arrangement is such that (e) is transcriptionallydownstream of (a) whilst (f) is transcriptionally downstream of (b).Here again (e) terminates the transcription initiated by (a), whilst (f)terminates the transcription initiated by promoter (b). Placement of theterminators outside of (d) allows the use of bi-directional terminatorsas well as uni-directional terminators, in contrast to the arrangementin FIG. 1( a) where uni-directional terminators are preferred because ofthe placement of the terminators between the promoters. A number ofbi-directional terminators which could be used in accordance with theinvention are known in the art. Generally these are observed to bepolymerase non-specific.

The embodiment shown in FIG. 1( b) provides essentially the sameadvantages as that shown in FIG. 1( a) over the prior art vectors fordsRNA production. The vector shown in FIG. 1( b) will lead to theproduction of transcripts which are slightly longer, including thepromoter regions. This relatively small difference in the length of thetranscript and hence the formed dsRNA will not severely affect theefficacy in an RNAi system.

The position of the terminators and promoter in the example as shown inFIG. 1( b) are preferably placed at close proximity, such that the 5′end of the promoters are separated from the 3′ end of the transcriptionterminators by no more than 2000 nucleotides, preferably no more than1000 nucleotides, more preferably no more than 500 nucleotides, evenmore preferably no more than 200 nucleotides, especially preferably nomore than 100 nucleotides, more especially preferably no more than 50nucleotides, even more especially preferably no more than 20nucleotides, particularly preferably no more than 10 nucleotides, moreparticularly preferably no more than 6 nucleotides.

FIG. 1( c) illustrates schematically a further DNA construct accordingto the invention which is a plasmid vector comprising two opposablepromoters (a) and (b) flanking an inter-promoter region (c). In thisembodiment one terminator (in this case (f)) is positioned within the(c) and the other (e) is positioned outside (c). Again, (e) terminatestranscription initiated by (a) and (f) terminates transcriptioninitiated by (b). This arrangement may provide a useful solution to theproblem of one of the terminators interfering with polymerase activityin the other direction (e.g. (f) interferes with (b) initiatedtranscription). This vector essentially provides the same advantages asthe vector variations shown in FIG. 1( a) and FIG. 1( b) over the priorart. The relatively small difference in the length of the transcript dueto the transcription of one of the promoters will not significantlyaffect the efficacy in RNAi systems. This more particularly the casewhen the terminator that is located outside of the inter-promoter region(c) is at close proximity of the promoter, as defined above.

FIGS. 1( d) and 1(e) illustrate schematically two further DNA constructsaccording to the invention which are both plasmid vectors comprising twoopposable promoters (a) and (b) flanking an inter-promoter region (c).These embodiments comprise a single terminator only. In the arrangementshown in FIG. 1( d) a single terminator (e) which terminatestranscription from (a) is placed outside of (c). The placement of theterminator outside of the inter-promoter region allows the use of both abi-directional terminator or a uni-directional terminator in thissystem. In the embodiment shown in FIG. 1( d), (e) is placed within the(c) and should therefore preferably be a uni-directional terminator.

Further embodiments of the DNA construct according to the invention areillustrated schematically in FIGS. 2( b) to 2(e).

These embodiments are all plasmid cloning vectors, based upon theoptimal arrangement of promoters and terminators shown in FIG. 1( a),and described above, containing cloning sites to facilitate theinsertion of a DNA fragment into the at least one cloning site.

These embodiments are all plasmid cloning vectors, based upon theoptimal arrangement of promoters and terminators shown in FIG. 1( a),containing cloning sites to facilitate the insertion of a target DNAfragment into the inter-promoter region.

FIG. 2( a), which is a schematic representation of a prior art cloningvector, is included for comparison purposes. This vector comprises twoopposable promoters (a) and (b), which may be identical or different,flanking a multi-cloning site (MCS).

FIG. 2( b) illustrates a first type of plasmid cloning vector accordingto the invention. The vector contains a first opposable promoter (a) anda second opposable promoter (b) flanking an inter-promoter region. Theinter-promoter region can further be defined as: the DNA region betweenthe 3′ end of the first promoter and the 3′ end of the second promoter,and which is downstream of the first promoter, and which is downstreamof the second promoter, and which preferably does not contain the 5′ endof the first promoter and of the second promoter. The inter-promoterregion further comprises terminators (e) and (f) flanking amulti-cloning site MCS. The MCS comprises at least one individualrestriction site, and preferably more than one restriction site as knownin the art, any of which may be used for insertion of a DNA fragment.

FIG. 2( c) illustrates a further type of plasmid cloning vectoraccording to the invention. This vector again contains opposablepromoters (a) and (b) flanking an inter-promoter region comprisingterminators (e) and (f). In this embodiment, (a) and (b) flank a cloningsite which is adapted for facilitated cloning of PCR fragments,comprising a stuffer DNA flanked by two identical restriction sites, inthis case BstXI sites. The specific sequence of the stuffer DNA is notessential, provided that said stuffer DNA does not interfere with thedesired effect and/or the desired activity of the DNA constructs of theinvention. A specific example of a vector according to this aspect ofthe invention described herein is plasmid pGN29.

The cloning of PCR products using BstXI recognition sites and BstXIadaptors is generally known in the art. The BstXI adaptors arecommercially obtained, such as from Invitrogen (Groningen, theNetherlands). These adaptors are non-palindromic adapters designed foreasier and efficient cloning of PCR products into vectors. These use ofthese adaptors reduces the concatamerization of adapters or insert DNAby having non-complementary (CACA) overhangs. The stuffer DNA isincluded merely to allow easy differentiation between BstXI cut anduncut vector on the basis of size. Its precise length and sequence arenot of importance.

FIG. 2( d) illustrates a further type of plasmid cloning vectoraccording to the invention. This vector again contains opposablepromoters (a) and (b) flanking an inter-promoter region comprisingterminators (e) and (f). In this embodiment, (a) and (b) flank a cloningsite which facilitates “High Throughput” cloning based on homologousrecombination rather than restriction enzyme digestion and ligation. Asshown schematically in FIG. 2( d), the cloning site comprises attR1 andattR2 recombination sites from bacteriophage lambda flanking a genewhich is lethal to E. coli, in this case the ccdB gene.

An alternative cloning method of DNA fragments into this vector,(notshown in FIG. 2( d)), consists of a variant of this vector, wherein theccdB DNA sequence further comprises at least one unique restrictionsite, preferably the at least unique restriction site is a blunt-endrestriction site, such as a SrfI restriction site. Insertion of a DNAfragment in the at least one unique restriction site, results ininactivation of the ccdB gene, and hence in inactivation of the lethalccdB gene product.

A further variant of a vector a shown in FIG. 2( d) in which the attR1and the attR2 are not present. Such a vector comprises at least onecloning site, said at least one cloning site consisting of a ccdBsequence, said ccdB sequence further comprising at least one uniquerestriction site, preferably the at least unique restriction site is ablunt-end restriction site, such as a SrfI restriction site. Insertionof a DNA fragment in the at least one unique restriction site, resultsin inactivation of the ccdB gene, and hence in inactivation of thelethal ccdB gene product.

These cloning sites comprising the ccdB nucleotide sequence and/or theattR sites (R1 and/or R2) are derived from the Gateway™ cloning systemcommercially available from Life Technologies, Inc. The Gateway™ cloningsystem has been extensively described by Hartley et al. in WO 96/40724(PCT/US96/10082). A specific example of a vector according to thisaspect of the invention described herein is pGN39.

FIGS. 2( e) and 2(f) illustrate a still further type of plasmid cloningvector according to the invention. This vector again contains opposablepromoters (a) and (b) flanking an inter-promoter region (c) comprisingterminators (e) and (f). In the embodiment shown in FIG. 2( e),terminators (e) and (f) flank a cloning site which facilitates “highthroughput” cloning of PCR products by TA™ cloning. This cloning sitecomprises a stuffer DNA flanked by two identical restriction sites foran enzyme which generates overhanging T nucleotides. In this case therestriction sites are XcmI sites, but other sites which are cleaved togenerate overhanging T nucleotides could be used with equivalent effect.The overhanging T nucleotides facilitate cloning of PCR products whichhave a overhanging A nucleotide. This principle is known as TA™ cloning.The cut vector with overhanging T nucleotides can be “topomerized” togenerate a cloning vector of the type shown schematically in FIG. 2( f),by linking the enzyme topoisomerase to the overhanging T nucleotides.The resulting vector also facilitates the cloning of PCR products by theprinciple known as TOPO™ cloning.

Both the TOPO™ and TA™ cloning systems, although not for the vectorsdescribed in this invention, are commercially available from Invitrogen.The TOPO™ cloning system has extensively been described by Shuman in WO96/19497 (PCT/US95/16099). The TA™ cloning system has extensively beendescribed by Hernstadt et al. in WO 92/06189 (PCT/US91/07147).

It will be readily appreciated by the skilled reader that whilst FIGS.2( b)-2(f) illustrate the inclusion of different cloning sites into avector of the type illustrated in FIG. 1( a), these cloning sites couldbe included into any of the DNA constructs of the invention, includingthose illustrated schematically in FIGS. 1( b) to 1(e)

Application of the DNA Constructs of the Invention in RNAi Technology.

As aforementioned, a major application of the DNA constructs/vectors ofthe invention is in the production of double stranded RNA for use inRNAi technology. In particular, the constructs are useful in in vivoRNAi protocols in the nematode worm C. elegans.

In C. elegans, RNAi has traditionally been performed by injection dsRNAinto the worm. Fire et al. describes these methods extensively inInternational Application No. WO 99/32619. In short, both strands of RNAare produced in vitro using commercially available in vitrotranscription kits. Both strands of RNA are allowed to form dsRNA, afterwhich the dsRNA is injected into C. elegans.

The new vector system developed by the present inventors is a drasticimprovement on this traditional method. First, the RNA can be producedin one step, for instance by using two identical promoters such as inthe vector pGN9. Second, and more important, due to the presence ofterminators the transcripts, and hence the formed dsRNA, will be morespecific as only the cloned target fragment will be transcribed. Thiswill result in a more efficient RNAi.

A further method to perform RNAi experiments in C. elegans has beendescribed by Plaetinck et al. in WO 00/01846. In this method C. elegansworms are fed with bacteria which produce dsRNA. The dsRNA passes thegut barrier of the worm and induces the same RNAi as if the dsRNA hasbeen injected. For these experiments, the preferred E. coli strain isHT115 (DE3), and the preferred C. elegans strain is nuc-1;gun-1. Theimproved vectors provided by the invention also improve the efficiencyof RNAi in this method, as shown in the example below, as only effectivedsRNA is produced.

Another method to perform RNAi has also been described by Plaetinck etal. in WO 00/01846. In short, this method is based on the production ofdsRNA in the worm itself. This can be done by using worm promoters inthe described vectors, or by using a transgenic worm expressing apolymerase specific for non-C. elegans promoters present in the vector,such that this polymerase drives transcription of the dsRNA. Thepromoters will preferentially be selected from the known bacteriophageRNA promoters, such as T7 or T3 or SP6 RNA promoters, which provide theadvantage of a high level of transcription dependent only on the bindingof the cognate polymerase.

Plasmid vector DNA can be introduced into the worm by several methods.First, the DNA can be introduced by the traditional injection method(Methods in Cell Biology, Vol 48, C. elegans Modern Biological Analysisof an organism, ed. by Epstein and Shakes). Second, the DNA can beintroduced by DNA feeding. As has been shown by Plaetinck et al. in WO00/01846, plasmid DNA can be introduced into the worm by feeding theworm with an E. coli strain that harbors the plasmid. Preferentially theE. coli strain is OP50, or MC1061 or HT115 (DE3) but any other strainwould suit for this purpose. The C. elegans strain is preferentially anuc-1 mutant strain or a nuc-1; gun-1 strain. The plasmid DNA from theE. coli passes the gut barrier and is introduced into the nematode,resulting in the expression of dsRNA. As with the other RNAi methodsdescribed above, the use of the new vector system will enhance the RNAiby the production of only specific dsRNA.

The invention will be further understood with reference to the followingexperimental Examples, together with FIGS. 3-17.

EXAMPLES Example 1 Vector Construction

The starting point for construction of the vectors exemplified hereinwas plasmid pGN1. This plasmid, described in the applicant's co-pendingInternational Application No. WO 00/01846, contains two opposable T7promoters flanking a multi-cloning site.

Vector construction was carried out according to standard molecularbiology techniques known in the art and described, for example, in F. M.Ausubel et al. (eds.), Current Protocols in Molecular Biology, JohnWiley & Sons, Inc. (1994).

1) Construction of pGN9

pGN1 was first digested with restriction enzymes EcoRI and KpnI.Oligonucleotides oGN27 (SEQ ID NO:3) and oGN28 (SE ID NO:4) (FIG. 7)were annealed to generate a double stranded fragment which was thenligated into the EcoRI/KpnI cut vector. The resulting plasmid wasre-digested with XbaI and HindIII. Oligonucleotides oGN29 (SEQ ID NO:5)and oGN30 (SEQ ID NO:6) were annealed to generate a double-strandedfragment which was then annealed into the XbaI/HindIII cut vector. Theresulting vector was designated pGN9 (FIGS. 4 and 10; SEQ ID NO:8).

2) Construction of Further Cloning Vectors

pGN29 (FIG. 9( a); FIG. 11; SEQ ID NO:9) was generated by replacing theMCS in pGN9 with a stuffer DNA flanked by BstXI sites. BstXI adaptersare commercially available from Invitrogen (Groningen, the Netherlands).

pGN39 (FIG. 9( b); FIG. 12; SEQ ID NO:39) was generated by followingsteps; pGN29 was digested with BsTXI. BstXI adapters (Invitrogen,Groningen, The Netherlands) were ligated to Cassette A provided by theGATEWAY™ system (Life Technologies, Inc.). Cassette A contains attR1,CmR, ccda, ccdB, attR2. The Cassette A with the adapters were thenligated into the digested pGN29, resulting in pGN39A. pGN39A contains aunique SrfI site in the ccdB gene.

The TopoRNAi vector (FIG. 9( c); FIG. 13; SEQ ID NO:11) was generated inthe following way; pGN29 was digested with BstXI. Using PCR with theprimers oGN103 (SEQ ID NO:14) and oGN104 (SEQ ID NO:15) and templatepCDM8 (Invitrogen, Groningen, The Netherlands), a stuffer was generated(SEQ ID NO: 20) which includes XcmI sites. Onto the PCR product, BstXIadapters were ligated, and the resulting ligation product was ligated inthe BstXI digested pGN29 vector resulting in the TopoRNAi vector.

oGN103 (SEQ ID NO:14): 5′ TACCAAGGCTAGCATGGTTTATCACTGATAAGTTGG 3′ oGN104(SEQ ID NO:15): 5′ TACCAAGGCTAGCATGGGCCTGCCTGAAGGCTGC 3′

pGN49A (SEQ ID NO:12) was constructed to insert an additional uniquenon-blunt restriction site and to delete the CmR gene from pGN39.Overlap PCR was used. A first PCR was performed with primers oGN126 (SEQID NO:16)and oGN127 (SEQ ID NO:17) and PGN39A as template. Using primersoGN128 (SEQ ID NO:18) and OGN129 (SEQ ID NO:19) and the same template asecond fragment was generated. Overlap PCR using the resulting fragmentsand primers oGN126P and oGN129P resulted in a final PCR product. To thisfinal PCR product, BstXI adapters were ligated, and the ligation productwas ligated into pGN29 digested with BstXI. The resulting vector wasdesignated pGN49A.

A control vector was generated to test the efficiency of the pGN49Acloning vector, such vector should contain the T7 promoters, but not theT7 terminators. For this, the XbaI insert of pGN49A was isolated andcloned in pGN1 digested with the same restriction enzyme. The resultingvector was designated pGN59A (SEQ ID NO:13).

oGN126 (SEQ ID NO:16): pGATCTGGATCCGGCTTACTAAAAGCCAGATAACAGTATGC oGN127(SEQ ID NO:17): GGAGACTTTATCGCTTAAGAGACGTGCACTGGCCAGGGGGATCACC oGN128(SEQ ID NO:18): CCAGTGCACGTCTCTTAAGCGATAAAGTCTCCCGTGAACTTTACCCGGTGGoGN129 (SEQ ID NO:19) pGCTGTGTATAAGGGAGCCTGACATTTATATTCCCCAG

Example 2 To Illustrate the Usefulness of the Improved Vectors in RNA.

This experiment was designed to illustrate the improved efficiency ofthe improved vectors of this invention in double-stranded RNAinhibition, as compared to the vectors known from the prior art. Asignificant increase on the efficacy of the system could be expected, asmore effective dsRNA was produced and hence RNAi performed better. Theexperimental system for this proof of concept experiment was to measureC. elegans rescue at 25° C. in nuc-1/pha-1(e2123)ts C. elegans mutantsby RNAi of sup35 using dsRNA feeding of pGN-2 (−terminator) and pGN-12(+terminator), with PGN-1 (empty vector) as a control and dilutor. Thepha-1 ts/sup-35 mutation has extensively been described by Schnabel inWO 99/49066.

The nuc-1 mutation in C. elegans provides for a C. elegans strainexhibiting better uptake abilities, such as the uptake of the dsRNAcomplexes than wild type C. elegans. This mutant is deleted in the majorDNAse enzymes, and inventors have proven in earlier co-pendingapplications that this C. elegans strain results in enhanced RNAi byfeeding the nematode with dsRNAs.

The pha-1(e2123)ts mutation provides a mutant C. elegans strain with aphenotype of survival at 15° C. and lethality at 25° C. This lethalityis suppressible by the inhibition of sup-35 expression. RNAi of sup-35should thus facilitate the rescue of pha-1(e2123)ts at 25° C. Thevectors of the present invention, when expressing dsRNA from sup-35,should increase the efficacy of sup-35 RNAi in rescuing pha-1(e2123)tsmutants at 25° C., compared to vectors that do not contain theterminators.

Vector pGN1 was used as empty vector. Vector pGN2 (−terminator) is avector harboring sup-35 DNA and expressing sup-35 dsRNA when introducedin the appropriate host, the vector has no transcriptional terminatorsinserted. Vector pGN12 (+terminator) is a vector as described above,containing the transcriptional terminators, and hence resulting inimproved dsRNA production when introduced into an appropriate host.Thus, this vector has two unidirectional transcriptional terminators,both placed inside of the inter-promoter region, and flanking the sup-35fragment. Use of the latter vector was expected to increase the efficacyof the system, here meaning a better rescue (survival) of pha-1(e2123)tsmutants at 25° C.

Experimental Conditions

12-well micro-titer plates were filled with approximately 2 ml of NGMagar per well. (1 liter of NGM agar: 15 g Agar, 1 g peptone, 3 g NaCl, 1ml cholesterol solution (5 mg/ml in EtOH), with sterile addition afterautoclaving of 9.5 ml 0.1M CaCl₂, 9.5 ml 0.1 ml MgSO₄, 25 ml 1MKH₂PO₄/K₂HPO₄ buffer pH 6, ampicillin (100 μg/l), 5 ml 0,1 M IPTG and 5ml nystatin solution (dissolved 10 mg/ml in 1:1 EtOH:CH₃COONH₄ 7.5 M)

The dried plates were spotted with approximately 50 μl of an overnightculture of bacteria HT115 (DE3) (Fire A, Carnegie Institution,Baltimore, Md.) transformed with the plasmids. Individual nematodes atthe L4 growth stage were then placed in single wells at day 1. In eachwell 1 nematode was placed (P1). At day two, the P1 nematodes wereplaced to a new well and left to incubate for a day. The same procedurewas repeated at day 3. All plates were further incubated at 25° C. toallow offspring to be formed. Sup35 RNAi induced survival (rescue) aswas measured by counting the offspring.

Results

RNAi experiment in C. elegans nuc-1/pha-1(e2123)ts mutants by feedingwith E. coli expressing sup-35 dsRNA.

Set Up:

-   pGN1 as control-   pGN2 (sup 35−Term.)    -   pGN12 (sup 35+Term.)-   pGN2+pGN1 dilutions ½, ¼, ⅛, 1/16, 1/32    -   pGN12+pGN1 dilutions ½, ¼, ⅛, 1/16, 1/32        Conditions:-   Incubation temperature 25° C.    Readout:

Count offspring (adult hermaphrodites) pGN1 (control) Day 1 0 0 0 0 Day2 0 0 0 0 Day 3 0 0 0 0 pGN2 (undiluted) pGN12 (undiluted) Day 1 12 4 4832 Day 1 16 29 37 14 Day 2 24 23 80 85 Day 2 27 22 57 2 Day 3 5 0 9 16Day 3 1 2 4 1 pGN 2 + 1, ½ dilution pGN 12 + 1, ½ dilution Day 1 0 7 0 2Day 1 22 28 103 61 Day 2 9 10 0 3 Day 2 36 45 53 40 Day 3 0 2 0 0 Day 33 3 25 1 pGN 2 + 1, ¼ dilution pGN 12 + 1, ¼ dilution Day 1 28 23 0 0Day 1 * 6 36 5 Day 2 6 3 0 0 Day 2 24 55 3 Day 3 0 0 0 0 Day 3 pGN 2 +1, ⅛ dilution pGN 12 + 1, ⅛ dilution Day 1 0 0 4 0 Day 1 31 12 16 38 Day2 0 0 11 0 Day 2 4 5 37 4 Day 3 0 0 0 0 Day 3 0 0 2 1 pGN 2 + 1, 1/16dilution pGN 12 + 1, 1/16 dilution Day 1 0 0 0 0 Day 1 1 0 0 0 Day 2 0 00 1 Day 2 2 0 0 1 little Day 3 0 0 0 0 Day 3 0 1 1 1 pGN 2 + 1, 1/32dilution pGN 12 + 1, 1/32 dilution Day 1 0 0 0 0 Day 1 0 0 1 0 Day 2 0 00 0 Day 2 0 L2 3 0 Day 3 0 0 0 0 Day 3 2 0 L3-L4 0 * mother diedConclusions

As expected, worms fed by bacteria harboring pGN1, did not result in theviable offspring, due to the lethal effect of the pha-1 mutation at thistemperature.

Feeding the nematodes with E. coli harboring pGN2 or pGN12 both resultin viable offspring. This is due to the feeding of the worm with dsRNAfrom sup-35. The remarkable difference between the two feedingexperiments can be seen in the dilution series. When diluting thebacteria harboring pGN2 with bacteria harboring pGN1, the number ofoffspring diminishes drastically, even at a low dilution of one to two.This dilution series indicates that high levels of dsRNA are needed tohave a proper RNAi induction. In the feeding experiment with bacteriaharboring pGN12, significant offspring is still observed at a dilutionof one to eight. This indicates that in the bacteria harboring pGN12,much more effective dsRNA is formed.

This experiment clearly shows that the addition of terminator sequencesin vectors to express dsRNA as described above provide a significantadvantage in the generation of RNAi.

Example 3 Comparison of RNAi Efficiency of Vectors with and without T7Terminators(pGN49 vs pGN59)

Three different genes have been cloned in the vectors pGN49A and pGN59A.The cloning was performed by amplifying the gene fragments with PfuI DNApolymerase producing blunt ends, facilitating cloning in these vectors.These PCR fragments were cloned into the vectors digested with SrfI.Correct fragment insertion of the clones was checked by PCR. Thefragments are chosen such that ds expression and RNAi results in alethal phenotype of the offspring. This procedure allows fast and easycomparison of the efficiency of the two vectors pGN49 and pGN59 in RNAi.

plasmid Gene (acedb) Vector backbone pGW5 B0511.8 pGN49A pGW9 C01G8.7pGN49A pGW11 C47B2.3 pGN49A pGW17 B0511.8 pGN59A pGW21 C01G8.7 pGN59ApGW23 C47B2.3 pGN59A

All the plasmids (pGW-series)are transformed in E. coli AB301-105 (DE3)bacteria by standard methodology. The bacteria are then grown in LB/ampat 37° C. for 14-18 h. These cultures were centrifuged and the bacterialpellet dissolved in S-complete buffer containing 1 mM IPTG and 100 μg/μlampicillin.

In 96 well plates containing 100 μl S-complete (containing 1 mM IPTG and100 μg/μl ampicillin final concentration) and 10 μl of bacteriasolution, 3 nematodes were added at each well, the nematodes were at theL1 growth stage.

The plates were incubated at 25° C. for 5-6 days. Each day the plateswere inspected for development of the larvae and the production of F1offspring.

Results

The RNAi was performed in eight-fold for each constructed plasmid. Theresults show that when T7 terminators are inserted into the vectorbackbone, the expected phenotype gives a 100% occurrence. When T7terminators are not used the reproducibility can decrease up to 50%. Asin the previous experiment, the results show that the addition ofterminators significantly enhances RNAi performance.

DNA fragment B0511.8 B0511.8 C01G8.7 C01G8.7 C47B2.3 C47B2.3 VectorpGN49A pGN59A PGN49A pGN59A pGN49A pGN59A Resulting PGW5 PGW17 PGW9PGW21 PGW11 PGW23 plasmid Percentage 100 75 100 87.5 100 50 lethalPercentage 0 25 0 12.5 0 50 offspring

PCR fragment generated by the primers oGN103 and oGN104 on templatepCDM8 (SEQ ID NO:20) taccaaggct agcatggttt atcactgata agttgg ataagttggtggacatatta tgtttatcag tgataaagtg tcaagcatga caaagttgca gccgaatacagtgatccgtg ccggccctgg actgttgaac gaggtcggcg tagacggtct gacgacacgcaaactggcgg aacggttggg ggtgcagcag ccggcgcttt actggcactt caggaacaagcgggcgctgc tcgacgcact ggccgaagcc atgctggcgg agaatcatac gcttcggtgccgagagccga cgacgactgg cgctcatttc tgatcgggaa tcccgcagct tcaggcaggcccatgctagc cttggtacca gcacaatgg

Overlap PCR Fragment, which was used to generate pGN49A (SEQ ID NO:21)gatctggatccggcttactaaaagccagataacagtatgcgtatttgcgcgctgatttttgcggtataagaatatatactgatatgtatacccgaagtatgtcaaaaagaggtgtgctatgaagcagcgtattacagtgacagttgacagcgacagctatcagttgctcaaggcatatatgatgtcaatatctccggtctggtaagcacaaccatgcagaatgaagcccgtcgtctgcgtgccgaacgctggaaagcggaaaatcaggaagggatggctgaggtcgcccggtttattgaaatgaacggctcttttgctgacgagaacagggactggtgaaatgcagtttaaggtttacacctataaaagagagagccgttatcgtctgtttgtggatgtacagagtgatattattgacacgcccgggcgacggatggtgatccccctggccagtgcacgtctcttaagcgataaagtctcccgtgaactttacccggtggtgcatatcggggatgaaagctggcgcatgatgaccaccgatatggccagtgtgccggtctccgttatcggggaagaagtggctgatctcagccaccgcgaaaatgacatcaaaaacgccattaacctgatgttctggggaatataaatgt caggctcccttatacacagc

Other aspects of the invention will be clear to the skilled artisan andneed not be repeated here. Each reference or publication cited herein isincorporated by reference in its entirety.

The terms and expressions which have been employed are used as terms ofdescription and not of limitation, and there is no intention in the useof such terms and expressions of excluding any equivalents of thefeatures shown and described or portions thereof, it being recognizedthat various modifications are possible within the scope of theinvention.

1. A DNA construct that produces double stranded RNA comprising: a) afirst promoter and b) a second promoter, in which the first and secondpromoter are in opposite orientation to each other and define: c) aninter-promoter region positioned downstream of the 3′ end of the firstpromoter and downstream of the 3′ end of the second promoter; and whichDNA construct further comprises: d) at least one cloning site positionedin the inter-promoter region; and e) a first transcription terminator,positioned, as seen from the 3′ end of the first promoter, downstream ofthe first promoter and downstream of the at least one cloning site,wherein the first transcription terminator is operably linked to thefirst promoter.
 2. A DNA construct according to claim 1, furthercomprising: f) a second transcription terminator positioned, as seenfrom the 3′ end of the second promoter, downstream of the secondpromoter and downstream of the at least one cloning site, wherein thesecond transcription terminator is operably linked to the secondpromoter.
 3. A DNA construct according to claim 1, in which the firsttranscription terminator is positioned in the inter-promoter region. 4.A DNA construct according to claim 1, in which the first transcriptionterminator is positioned, as seen from the 3′ end of the first promoter,downstream of the first promoter, downstream of the at least one cloningsite, and downstream of the 5′ end of the second promoter.
 5. A DNAconstruct according to claim 2, in which the second transcriptionterminator is positioned in the inter-promoter region.
 6. A DNAconstruct according to claim 2, in which the second transcriptionterminator is positioned, as seen from the 3′ end of the secondpromoter, downstream of the second promoter, downstream of the at leastone cloning site, and downstream of the 5′ end of the first promoter. 7.A DNA construct according to claim 4, in which the 3′ end of the firsttranscription terminator is separated from the 5′ end of the secondpromoter by no more than 2000 nucleotides.
 8. A DNA construct accordingto claim 7, in which the 3′ end of the second transcription terminatoris separated from the 5′ end of the first promoter by no more than 500nucleotides.
 9. A DNA construct according to claim 1 wherein the firstand the second promoter are identical.
 10. A DNA construct according toclaim 1 wherein the first and the second promoter are non-identical. 11.A DNA construct according to claim 8 wherein the first promoter and thesecond promoter are independently chosen from T7, T3 or SP6 promoters.12. A construct according to claim 1 wherein the cloning site comprisesat least one restriction site.
 13. A DNA according to claim 12 whereinthe cloning site comprises at least two restriction sites flanking asequence of DNA.
 14. A DNA construct according to claim 13 wherein theat least two restriction sites are identical.
 15. A DNA constructaccording to claim 12 wherein the at least one restriction site areBstXI sites.
 16. A DNA construct according to claim 12 wherein the atleast one restriction sites are XcmI sites.
 17. A DNA constructaccording to claim 1 wherein the cloning site further comprises attR1and attR2 recombination sequences.
 18. A DNA construct according toclaim 1 wherein the cloning site further comprises a ccdB nucleotidesequence.
 19. A DNA construct according to claim 18 wherein the ccdBnucleotide sequence further comprises at least one unique restrictionsite.
 20. A DNA construct according to claim 19 wherein the at least oneunique restriction site is at least one blunt-end restriction site. 21.A DNA construct according to claim 20 wherein the at least one blunt-endrestriction site is at least one SrfI site.
 22. A DNA according to claim2 which further comprises: (g) a DNA fragment inserted in the at leastone cloning site.
 23. A DNA construct according to claim 1 which is aplasmid or vector.
 24. A DNA construct according to claim 7, in whichthe 3′ end of the second transcription terminator is separated from the5′ end of the first promoter by no more than 200 nucleotides.
 25. Amethod for the production of double-stranded RNA for RNA inhibitioncomprising using the DNA construct of any of claims 1-23 or claim 24 forthe expression of double stranded RNA inserted in the cloning site. 26.A bacterial strain harbouring the DNA construct according to claim 1.27. A bacterial strain according to claim 26, wherein said bacterialstrain is an E. coli strain.
 28. A method for the production ofdouble-stranded RNA for RNA inhibition comprising using the bacterialstrain of claim 26 or claim 27 for the expression of double stranded RNAinserted in the cloning site.